NEXT SCHEDULED UPDATE 31 December 2007 No. unique visitors = 52,816 up to 27 March 2005 (2062 days) + Scroll Down OR Jump to Latest Entry.
April 2006 "Pain Relief Drugs from the Sea"
Analgesic Component of Venom (ACV1) from Cone Snails :
For a one-page description of Cone Shells and their Conotoxins click here For a video simulation of cone shell envenomaton click here
Bruce Livett's more recent publications (1998-2006)
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Live Cone Snails from the Seychelles
David Touitou has posted some amazing photos of live cone shells (some on eggs !!) taken during his November 2007 trip to the Seychelles (family trip). Included are photos of the following Conus gubernator leehmani, Conus canonicus, Conus episcopatus, Conus omria, Conus pennaceus, Conus legatus . You can find these on his homepage http://www.seashell-collector.com/index.htm
26 December, 2007
The Conidae of South Africa
ConchBooks have announced a forthcoming book in progress for release in 2008. The Conidae of South Africa by Manuel Tenorio, part 15 in the series A Conchological Iconography.
Intrathecal Ziconotide trial for treatment of pain
Ellis DJ, Dissanayake S, McGuire D, Charapata SG, Staats PS, Wallace MS, Grove GW, Vercruysse, P and the Elan Study 95-002 Group (2008) Continuous intrathecal infusion of Ziconotide for treatment of chronic malignant and nonmalignant pain over 12 months: A prospective, open-label study. Neuromodulation 11: 40-49.
Abstract: Objectives. This study aims to assess the safety and efficacy of long-term intrathecal (IT) ziconotide infusion.
17 December, 2007
Internet Hawaiian Shell News (IHSN)
The IHSN is an Internet Magazine that replaced the printed Hawaiian Shell News (HSN) in January 1997. Both are published by Hawaiian Malacological Society. The new IHSN Portal URL (on 1&1 Web Hosting), on which all issues of IHSN from 1996 to date are accessible is http://s190418054.onlinehome.us/index.html.
12 December, 2007
New postage stamp depicting a cone shell
During 2007, a new postage stamp depicting Conus clenchi was released by Uruguay. Tom Walker again kindly provided me with the image. It has been added to the Cone Shells on Stamps resource and is available for viewing by clicking here.
5 December, 2007
Nonclinical safety of Ziconotide
Skov MJ,a Beck JC,b de Kater AWc and Shopp GM,d (2007) Nonclinical safety of Ziconotide: An intrathecal analgesic of a new pharmaceutical class. Int. J. Toxicol. 26: 411-421.
Abstract: Ziconotide, a potent, selective, reversible blocker of neuronal N-type voltage-sensitive calcium channels, is approved in the United States for the management of severe chronic pain in patients for whom intrathecal therapy is warranted, and who are intolerant or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. In the European Union, ziconotide is indicated for the treatment of severe chronic pain in patients who require intrathecal analgesia. Nonclinical investigations of ziconotide included a comprehensive characterization of its toxicology, incorporating acute and subchronic toxicity studies in rats, dogs, and monkeys; reproductive toxicity assessments in rats and rabbits; and mutagenic, carcinogenic evaluations performed in vivo and in vitro. Additional investigations assessed the potential for cardiotoxicity (rats) and immunogenicity (mice, rats, and guinea pigs), and the presence or absence of intraspinal granuloma formation and local cell proliferation and apoptosis (dogs). The resulting nonclinical toxicology profile was predictive of human adverse events reported in clinical trials and consistent with ziconotide's pharmacological activity. Frequently observed nonclinical behavioral effects included tremoring, shaking, ataxia, and hyperreactivity. Occurrences were generally transient and reversible upon cessation of treatment, and intolerable effects occurred at doses more than 45 times the maximum recommended clinical dose. Ziconotide was not associated with target organ toxicity, teratogenicity, or treatment-related gross or histopathological changes; it displayed no mutagenic or carcinogenic potential and no propensity to induce local cell proliferation or apoptosis. Although guinea pigs developed systemic anaphylaxis, antibodies to ziconotide were not detected in mice, rats, or guinea pigs, indicating low immunogenic potential. No evidence of granuloma formation was observed with intrathecal ziconotide treatment. In summary, the results from these nonclinical safety assessments revealed no significant toxicological risk to humans treated with ziconotide as recommended.
Solid-phase microwave assisted synthesis of Conotoxin MII
Galanis AS, Albericio F and Grotli M (2007)Development of new synthetic strategies for synthesis of α-conotoxin-MII.
Proc. 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece
August 20–25, 2007, Amino Acids 33 (3) pp. XIV-XV.
Abstract: Conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup a-conotoxins specifically and selectively bind to subtypes of nicotinic acetylcholine receptors (nAchRs), which are targets for treatment of several neurological disorders. Until now, conotoxins have been mainly prepared by Solid Phase Peptide Synthesis (SPPS) combined with formation of the disulfide bridges after releasing the linear conotoxin precursor from the solid support. We have developed a solid-phase microwave assisted synthesis strategy for the preparation of a-conotoxins.
Structural basis for high specificity binding of the J-superfamily conotoxin pl14a to the Kv1.6 channel
Mondal S and Ramakumar S (2007) Computational analysis of sequences and structures of conotoxins. Proc. 10th International Congress on Amino Acids and Proteins (ICAAP), Kallithea, Greece August 20–25, 2007, Amino Acids 33 (3) p. X.
Abstract: Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors.
Characterizing disulfide-bridged peptides in Conus textile venom
Quinton L, Demeure K, Dobson R, Gilles N, Gabelica V, De Pauw E. (2007) New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms. J Proteome Res. 6: 3216-3223.
Abstract: Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.
Biofutur N° 280: Les venins, nouvelles sources de médicaments?
2 December, 2007
α4/3 Conotoxins - Review
Ellison M and Olivera BM (2007). alpha4/3 Conotoxins: phylogenetic distribution, functional properties, and structure-function insights. Chem Rec. 2007 Nov 29;7(6):341-353 [Epub ahead of print]
Abstract: This review examines the alpha4/3 conotoxins as an example of molecular diversity in a class of compounds that have evolved in a group of closely related species in a single phylogenetic lineage. The species examined belong to Stephanoconus, a clade of Conus, a genus that contains 500-700 different species of carnivorous marine snails. We examine earlier work that describes the identification and characterization of alpha-ImI, the founding alpha4/3 toxin, and two other alpha4/3 toxins, alpha-ImII and alpha-RgIA. These three toxins all inhibit nicotinic acetylcholine receptors (nAChRs) belonging to a subset of nAChRs that are composed of only alpha subunits; they are, however, diverse in terms of the all-alpha subtype they preferentially antagonize and the receptor site that they bind to. We thus speculate that the alpha4/3 toxins may be a rich source of functionally diverse all-alpha subunit nAChR inhibitors. We review extensive work that has established a detailed model for alpha-ImI binding to one of its preferred nAChR subtypes (the alpha7 nAChR) and, by comparing the alpha-ImI, alpha-ImII and alpha-RgIA sequences demonstrate how structural features of alpha4/3 peptides that account for their diverse functional properties can be identified. This approach is extended to derive models of receptor-toxin binding that may account for the different subtype specificities of alpha4/3 peptides. We also speculate on how rational modification of alpha4/3 toxins may allow engineering of ligands with desired subtype specificities. The chemical diversity produced by the closely related animals in Stephanoconus is thus functionally differentiated, although structurally homologous. (c) 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 7: 341-353; 2007: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20131.
Ziconotide (ω-conotoxin MVIIA)- A Case Report
Valia-Vera JC, Villanueva VL, Asensio-Samper JM, Lopez-Alarcon MD and de Andres
JA (2007). [Ziconotide: an innovative alternative for intense chronic neuropathic pain.]
Rev Neurol. 45:665-669. Spanish.
Abstract: INTRODUCTION. Intense chronic pain is a very important health problem, as it has a high prevalence (5-10%), a multifactorial aetiology and its management is very often a very complex affair. Treatment of severe cases sometimes requires interventional approaches, such as continuous intrathecal infusion of opioids. CASE REPORT. We report the case of a 38-year-old female with intense neuropathic pain in the lower back and the lower limbs secondary to three operations on the L5-S1 lumbar segment. After implementing several different pharmacological regimes involving both oral and implanted systems (spinal cord stimulation and subarachnoid infusion pump with different pharmacological combinations) with no clinical improvement, intrathecal infusion with ziconotide was included in the protocol. CONCLUSIONS. Ziconotide is the first specific neuronal blocker that acts on the calcium channel by blocking the N-type voltage-dependent calcium channels. It is a new non-opioid analgesic with approved indication in the treatment of intense chronic pain, in patients who require intrathecal analgesics and are refractory to other analgesic treatments. Therefore, we shall have to consider this drug as a therapeutic alternative in patients do not experience sufficient relief with the pharmacological agents and means currently available to treat them.
1 December, 2007
Research on Conopeptides as Analgesics
Richard Lewis, Paul Alewood, David Adams and MacDonald Christie are Chief Investigators in this NHMRC funded Research Program Dissecting Pain Pathways using Venom Peptides. This is a joint project between the Institute for Molecular Bioscience and The School of Biomedical Sciences at the University of Queensland and the Pain Management Research Institute at the University of Sydney.
"A major aim of this project is to identify and characterise novel venom peptides from Australia’s venomous creatures, especially the cone snails, which may hold therapeutic potential".
Download a review of venom peptides and their potential as therapeutics:
29 November, 2007
Animal toxins for pain control - Review
Cury Y and Picolo G. (2006) Animal toxins as analgesics--an overview. Drug News Perspect. 19: 381-392. Review.
Abstract: Pain is a multidimensional sensory experience, and multiple mechanisms are involved in the generation of pathophysiological nociceptive pain. Identification of the mechanisms and molecular components responsible for pain generation has not only advanced our understanding of pain and its control, but has also led to the selection of new targets for designing novel analgesic drugs. The high selectivity and specificity of animal toxins have enabled their use as potential therapeutics in the treatment of pain and candidates for the development of new analgesic drugs. This review focuses on the use of animal toxins for pain control and examines the possible analgesic mechanisms of these molecules. Copyright (c) 2006 Prous Science. All rights reserved.
25 November, 2007
Tenorio MJ, Poppe GT & Tagaro SP (2007) (n. Sp. and ssp. Of Conus)
VISAYA 2: (2) November 2007 [Malacological Journal of Conchology, Inc., Cebu, Philippines] Features images of Conus acutangulus Lamarck, 1810 Philippines ; Conus beatrix n. sp. Northern Territory, Darwin, Australia; Conus pagodus Kiener, 1845 Philippines; and Conus praecellens A. Adams, 1854 Philippines (see Plate 4, p. 90).
New species of Conus described in VISAYA include Conus frausseni, Conus grohi, Conus terryni (vol 1 no.1); Conus medoci, Conus chiapponorum, Conus vulcanus, Conus claudiae, Conus isabelarum, Conus crioulus, Conus suduirauti (Vol 1 no.2); Conus betulinus rufoluteus (Vol.1 no. 4); Conus guidopoppei (Vol.1 no.5) and Conus beatrix (Vol. 2 no.2). For more information visit: http://www.conchology.be/en/shelltopics/visaya/
α-Conotoxin MII used to detect α-6 nicotinic receptors in the brain
Exley R, Clements MA, Hartung H, McIntosh JM and Cragg SJ (2007). α6-Containing Nicotinic Acetylcholine Receptors Dominate the Nicotine Control of Dopamine Neurotransmission in Nucleus Accumbens. Neuropsychopharmacology. 2007 Nov 21; [Epub ahead of print]
Abstract: Modulation of striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Nicotine, by
desensitizing β2 subunit-containing (β2(*)) nicotinic acetylcholine receptors (nAChRs) on striatal DA axons, significantly enhances how DA is released by reward-related burst activity compared to nonreward-related tonic activity. This action provides a synaptic mechanism for nicotine to facilitate the DA-dependent reinforcement. The subfamily of β2(*)-nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of α6β2(*)-nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real-time using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in mouse striatal slices. The α6-specific antagonist α-conotoxin-MII suppressed DA release evoked by single and low-frequency action potentials and concurrently enhanced release by high-frequency bursts in a manner similar to the β2(*)-selective antagonist dihydro-beta-erythroidine (DHbetaE) in NAc, but less so in CPu. The greater role for α6(*)-nAChRs in NAc was not due to any confounding regional difference in ACh tone since elevated ACh levels (after the acetylcholinesterase inhibitor ambenonium) had similar outcomes in NAc and CPu. Rather, there appear to be underlying differences in nAChR subtype function in NAc and CPu. In summary, we reveal that α6β2(*)-nAChRs dominate the effects of nicotine on DA release in NAc, whereas in CPu their role is minor alongside other β2(*)-nAChRs (eg α4(*)), These data offer new insights to suggest striatal α6(*)-nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.
20 November, 2007
Knotted conopeptides !
Gracy J1,2, Le-Nguyen D3, Gelly J-C4, Kaas Q5, Heitz A1,2 and Chiche L1,2 (2007) KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007
Nucleic Acids Res. Open Access 10.1093/nar/gkm939 - published 19 November 2007, .
Abstract: The KNOTTIN database provides standardized information on the small disulfide-rich proteins with a knotted topology called knottins or inhibitor cystine knots. Static pages present the essential historical or recent results about knottin discoveries, sequences, structures, syntheses, folding, functions, applications and bibliography. New tools, KNOTER3D and KNOTER1D, are provided to determine or predict if a user query (3D structure or sequence) is a knottin. These tools are now used to automate the database update. All knottin structures and sequences in the database are now standardized according to the knottin nomenclature based on loop lengths between knotted cysteines, and to the knottin numbering scheme. Therefore, the whole KNOTTIN database (sequences and structures) can now be searched using loop lengths, in addition to keyword and sequence (BLAST, HMMER) searches. Renumbered and structurally fitted knottin PDB files are available for download as well as renumbered sequences, sequence alignments and logos. The knottin numbering scheme is used for automatic drawing of standardized two-dimensional Colliers de Perles of any knottin structure or sequence in the database or provided by the user. The KNOTTIN database is available at http://knottin.cbs.cnrs.fr.
Information about folding of conotoxins is here
Parallels between snake dendrotoxins and cone snail conkunitzins
Imperial JS, Silverton N, Olivera BM, Bandyopadhyay PK, Sporning A, Ferber M and Terlau H. (2007) On the origins of fish-hunting in venomous cone snails. Proc. Am. Phil. Soc. 151: 185-200.
14 November, 2007
Predicting d-amino acids in conotoxins
Buczek O, Jimenez EC, Yoshikami D, Imperial JS, Watkins M, Morrison A and Olivera
BM (2007). I(1)-superfamily conotoxins and prediction of single d-amino acid occurrence.
Toxicon. 2007 Sep 29; [Epub ahead of print]
Abstract: The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.
PrIIIE, a novel conotoxin from Conus parius
Lluisma AO, López-Vera E, Bulaj G, Watkins M and Olivera BM (2007) Characterization of a novel ψ-conotoxin from Conus parius, Reeve. Toxicon (2007), doi:10.1016/j.toxicon.2007.07.009
Abstract: The M-superfamily of conotoxins currently comprises three major groups of peptides (the μ-, κM-, and ψ-families) that share a key structural characteristic, the six-Cysteine motif CC-C-C-CC, but differ with respect to their molecular targets. The ψ-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two ψ-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, C. purpurascens. In this paper, we report the discovery and initial characterization of a ψ-conotoxin from another Conus species, C. parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmole, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmole of peptide) and did not block action potential in directly-stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC50 ~ 250 nM) was significantly more potent than PIIIE (IC50 ~ 7000 nM) and that PrIIIE showed higher ihhibition potency against the adult-type than the fetal type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na+ channel isoform Nav 1.4. The discovery of this ψ-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of ψ-conotoxins remains to be discovered from the members of this subgenus.
Functional similarity between DSP and conotoxin GVIA
Kouno T, Mizuguchi M, Tanaka H, Yang P, Mori Y, Shinoda H, Unoki K, Aizawa T,
Demura M, Suzuki K, Kawano K. (2007) The Structure of a Novel Insect Peptide Explains Its Ca(2+) Channel Blocking and Antifungal Activities. Biochemistry. 2007 Nov 10; [Epub ahead of print]
Abstract: Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.
8 November, 2007
"Tying the knot": Cyclic cystine knot scaffolds
Cemazar M, Gruber CW, Craik DJ (2007). Oxidative Folding of Cyclic Cystine Knot Proteins. Antioxid Redox Signal. Oct 25; [Epub ahead of print]
Abstract: Cyclic cystine knot proteins are small but topologically complex molecules that occur naturally in plants and have a wide range of bioactivities that make them interesting from a pharmaceutical perspective. Their remarkable stability is dependent on the correct formation of a knotted arrangement of disulfide bonds. This review reports on studies that have deciphered the pathways to the "tying of the knot." These studies have involved a range of biophysical techniques and suggest that the major intermediate species presented on these pathways are two disulfide native species, which are not necessarily the precursors of the native protein. Structural elucidations of one analogue and one such intermediate have been reported, and they both show highly native-like conformation and native disulfide bond connectivity. Cyclic cystine knot formation has also been shown to be assisted by protein disulfide isomerase. The points summarized in this review will be important to consider in the design of novel pharmaceutically interesting biomolecules based on the cyclic cystine knot motif, which has shown potential as a molecular scaffold because of its exceptional stability.
See also : Gruber CW, Cemazar M, Heras B, Martin JL, and Craik DJ. (2006) Protein disulfide isomerase: the structure of oxidative folding. Trends
Biochem Sci 31: 455–464.
6 November, 2007
CyBase: database of cyclic protein sequences
Abstract: CyBase was originally developed as a database for backbone-cyclized proteins, providing search and display capabilities for sequence, structure and function data. Cyclic proteins are interesting because, compared to conventional proteins, they have increased stability and enhanced binding affinity and therefore can potentially be developed as protein drugs. The new CyBase release features a redesigned interface and internal architecture to improve user-interactivity, collates double the amount of data compared to the initial release, and hosts a novel suite of tools that are useful for the visualization, characterization and engineering of cyclic proteins. These tools comprise sequence/structure 2D representations, a summary of grafting and mutation studies of synthetic analogues, a study of N- to C-terminal distances in known protein structures and a structural modelling tool to predict the best linker length to cyclize a protein. These updates are useful because they have the potential to help accelerate the discovery of naturally occurring cyclic proteins and the engineering of cyclic protein drugs. The new release of CyBase is available at http://research1t.imb.uq.edu.au/cybase [Using the cyclization tools incorporated into CyBase, Fig. 2(B) in this article shows a model of a cyclic alpha conotoxin MII using its native linear structure as a template (PDB ID: 1MII) which has been cyclized in silico using a seven-residue poly-alanine linker.
Prialt (omega-conotoxin MVIIA) elevated to first-line alternative for intraspinal pain treatment
Deer, T. et al (2007) Polyanalgesic Consensus Conference 2007: Recommendations for the Management of Pain by Intrathecal (Intraspinal) Drug Delivery: Report of an Interdisciplinary Expert Panel Neuromodulation 10: 300–328.
Abstract:
Press Release: CHARLESTON, W.Va., Oct. 31 /PRNewswire/ -- The 2007 Polyanalgesic Consensus Panel (PCP) -- a group of leading national pain management physicians from the United States and abroad -- has updated their intraspinal pain treatment guidelines and recommendations. The findings were released this month in Neuromodulation, a neurology and pain publication for physicians. The panel of pain experts revised the guidelines used to determine treatment via intraspinal infusion for patients suffering from severe chronic pain. The updated algorithm includes Ziconotide (PRIALT Elan Corp.) as a first-line alternative for intraspinal infusion to the opioids morphine and hydromorphone.
Read report here.
[Dr. Baldomero M. Olivera, who's research on cone shells and conotoxins led to the discovery of omega-conotoxin MVIIA and the development of Prialt, was named 2007 Scientist of the Year by the Harvard Foundation at Harvard University, and will receive the degree of doctor of science honoris causa from the University of the Philippines on Jan. 16, 2008. Read news release here ]
5 November, 2007
Radulae of Conus from India
Franklin JB,1,3, Fernando SA,1 Chalke BA,2 and Krishnan KS,2,3* (2007) Radular morphology of Conus (Gastropoda: Caenogastropoda: Conidae) from India. Molluscan Research 27: 111-122.
Abstract: Radular morphologies of 22 species of the genus Conus from Indian coastal waters were analyzed by optical and scanning electron microscopy. Although the majority of species in the present study are vermivorous, all three feeding modes known to occur in the genus are represented. Specific radular-tooth structures consistently define feeding modes. Species showing similar feeding modes also show fine differences in radular structures. We propose that these structures will be of value in species identification in cases of ambiguity in other characteristics. Examination of eight discrete radular-tooth components has allowed us to classify the studied species of Conus into three groups. We see much greater inter-specific differences amongst vermivorous than amongst molluscivorous and piscivorous species. We have used these differences to provide a formula for species identification. The radular teeth of Conus araneosus, C. augur, C. bayani, C. biliosus, C. hyaena, C. lentiginosus, C. loroisii, and C. malacanus are illustrated for the first time. In a few cases our study has also enabled the correction of some erroneous descriptions in the literature.
Disulfide isomerase from Conus amadis
Gowd KH, Krishnan KS and Balaram P (2007). Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation. Mol Biosyst. 3(8):554-566.
Abstract: Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.
1 November, 2007
Mitochondrial genome of Conus textile
Bandyopadhyay PK, Stevenson BJ, Ownby JP, Cady MT, Watkins M and Olivera BM (2007).
The mitochondrial genome of Conus textile, coxI-coxII intergenic sequences and
Conoidean evolution. Mol Phylogenet Evol. 2007 Aug 24; [Epub ahead of print]
Abstract: The cone snails belong to the superfamily Conoidea, comprising approximately 10,000 venomous marine gastropods. We determined the complete mitochondrial DNA sequence of Conus textile. The gene order is identical in Conus textile, Lophiotoma cerithiformis (another Conoidean gastropod), and the neogastropod Ilyanassa obsoleta, (not in the superfamily Conoidea). However, the intergenic interval between the coxI and coxII genes was much longer in C. textile(165bp) than in any other previously analyzed gastropod. We used the intergenic region to evaluate evolutionary patterns. In most neogastropods and three conidean families the intergenic interval is small (<30 nucleotides). Within Conus, the variation is from 130 to 170bp, and each different clade within Conus, has a narrower size distribution. In Conasprella, a subgenus traditionally assigned to Conus, the intergenic regions vary between 200 and 500bp, suggesting that the species in Conasprella are not congeneric with Conus. The intergenic region was used for phylogenetic analysis of a group of fish-hunting Conus, despite the short length resolution was better than using standard markers. Thus, the coxI-coxII intergenic region can be used both to define evolutionary relationships between species in a clade, and to understand broad evolutionary patterns across the large superfamily Conoidea.
Terebridae, not Conus ! but venomous nevertheless
Imperial JS, Kantor Y, Watkins M, Heralde FM III, Stevenson B, Chen P, Hansson
K, Stenflo J, Ownby JP, Bouchet P and Olivera BM. Venomous auger snail Hastula (Impages) hectica (Linnaeus, 1758): molecular phylogeny, foregut anatomy and comparative toxinology. J. Exp. Zool. (Mol. Dev. Evol.) 308B:744–756
Abstract: The >10,000 living venomous marine snail species [superfamily Conoidea (Fleming, 1822)] include cone snails (Conus), the overwhelming focus of research. Hastula hectica (Linnaeus, 1758), a venomous snail in the family Terebridae (Mörch, 1852) was comprehensively investigated. The Terebridae comprise a major monophyletic group within Conoidea. H. hectica has a striking radular tooth to inject venom that looks like a perforated spear; in Conus, the tooth looks like a hypodermic needle. H. hectica venom contains a large complement of small disulfide-rich peptides, but with no apparent overlap with Conus in gene superfamilies expressed. Although Conus peptide toxins are densely post-translationally modified, no post-translationally modified amino acids were found in any Hastula venom peptide. The results suggest that different major lineages of venomous molluscs have strikingly divergent toxinological and venom-delivery strategies.
28 October, 2007
Folding of conotoxins: disulfide bridge formation
Bulaj G and Olivera BM (2007). Folding of conotoxins: Formation of the native disulfide bridges during chemical synthesis and biosynthesis of Conus peptides. Antioxid Redox Signal. 2007 Oct 25; [Epub ahead of print]
Abstract: Conopeptides from >700 species of predatory marine Conus snails provide an impressive molecular diversity of cysteine-rich peptides. Most of the estimated 50,000-100,000 distinct conopeptides range in size from 10 to 50 amino acid residues, often with multiple posttranslational modifications. The great majority contain from two to four disulfide bridges. As the biosynthetic and chemical production of this impressive repertoire of disulfide-rich peptides has been investigated, particularly the formation of native disulfide bridges, differences between in vivo and in vitro oxidative folding have become increasingly evident. In this article, we provide an overview of the molecular diversity of conotoxins with an emphasis on the cysteine patterns and disulfide frameworks. The conotoxin folding studies reviewed include regioselective and direct oxidation strategies, recombinant expression, optimization of folding methods, mechanisms of in vitro folding, and preliminary data on the biosynthesis of conotoxins in venom ducts. Despite these studies, how the cone snails efficiently produce properly folded conotoxins remains unanswered. As chemists continue to master oxidative folding techniques, insights gleaned from how conotoxins are folded in vivo will likely lead to the development of the new folding methods, as well as shed some light on fundamental mechanisms relevant to the protein folding problem.
Conantokins from Conus parius
Teichert RW, Jimenez EC, Twede V, Watkins M, Hollmann M, Bulaj G, Olivera BM (2007).
Novel conantokins from Conus parius venom are specific antagonists of NMDA receptors.
J Biol Chem. 2007 Oct 25; [Epub ahead of print]
Abstract: We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2 and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a posttranslationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the third position from the N-terminus, where the Gla residue is replaced by either aspartate or by another posttranslationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different posttranslational modifications. Conantokins-Pr1 and -Pr2 adopt a-helical conformations in the presence of divalent cations (Mg2+ and Ca2+), but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-D-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2 and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.
25 October, 2007
Conotoxins that interact with sodium channels
Ekberg J, Craik DJ and Adams DJ (2007). Conotoxin modulation of voltage-gated sodium channels.Int J Biochem Cell Biol. 2007 Sep 14; [Epub ahead of print]
Abstract: The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.
21 October, 2007
4th Int. Peptide Symposium, Cairns, 21-25 October , 2007
The Scientific Program for the 4th Int. Peptide Symposium held in Cairns, 21-25 October , 2007 includes the following conopeptide-related presentations:
Abstracts:
ConoServer - an online Database
David Craik and his research team from the University of Queensland have created a database from over 2000 published sequences of peptides from cone snails. This resource called ConoServer provides a comprehensive overview of conopeptide diversity and their uses as drugs, drug leads and diagnostic tools. Well organized and easily navigable, it can be accessed at http:research1t.imb.uq.edu.au/conoserver/
T-1 Conotoxin lt5d from Conus litteratus
Liu J, Wu Q, Pi C, Zhao Y, Zhou M, Wang L, Chen S, Xu A, (2007) Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels. Peptides (2007), doi:10.1016/j.peptides.2007.09.006
Abstract: A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous-hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole-cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC50 value of 156.16nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.
17 October, 2007
T-1 Conotoxins Pu5.1-Pu5.6 from Conus pulicarius
Peng C, Wu X, Han Y, Yuan D, Chi C, Wang C. (2007) Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning. Peptides 2007 Sep 4; [Epub ahead of print]
Abstract: Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a
3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.
Conlysin-Mt a cytolytic peptide from Conus mustelinus
Biggs JS, Rosenfeld Y, Shai Y and Olivera BM. (2007) Conolysin-Mt : A Conus peptide that disrupts cellular membranes. Biochemistry 2007 Oct 10; [Epub ahead of print]
Abstract: Conus venoms are estimated to comprise over 100,000 distinct pharmacologically
active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 muM, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC:cholesterol) model membranes. The affinity constants (KA) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 107 and 3 +/- 1 x 107 M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 muM) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 muM. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.
I-Superfamily Conotoxin i-RXIA (r11a)
Buczek BO, Wei D, Babon JJ, Yang X, Fiedler B, Chen P, Yoshikami D, Olivera
BM, Bulaj G, Norton RS.(2007) Structure and Sodium Channel Activity of an Excitatory I(1)-Superfamily Conotoxin.Biochemistry. 46: 9929-9940
Abstract: Conotoxin i-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a -C-C-CC-CC-C-C- pattern. i-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is posttranslationally isomerized to the D configuration. Naturally occurring i-RXIA with D-Phe44 is significantly more active as an excitotoxin than the L-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that i-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the D-Phe44 and L-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of i-RXIA as a voltage-gated sodium channel; i-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in i-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.
10 October, 2007
20,000 Ligands under the Sea ! A Review
Olivera BM and Teichert RW (2007) Diversity of the Neurotoxic Conus Peptides. A Model for Concerted Pharmacological Discovery. Review. Molecular Interventions 7:251-260, (2007)
Flourescent alpha6 and beta3 nAChRs
Drenan MR, Nashmi E, Imoukhuede PI, Just H, McKinney S and Lester HA (2007) Subcellular Trafficking, Pentameric Assembly and Subunit Stoichiometry of Neuronal Nicotinic ACh Receptors Containing Fluorescently-Labeled {alpha}6 and {beta}3 Subunits. Molecular Pharmacology (Accepted 11 October, 2007)
Abstract: Neuronal nicotinic ACh receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing {alpha}4, {alpha}6, {beta}2, and {beta}3 subunits are expressed in midbrain dopaminergic neurons and are implicated in the response to smoked nicotine. Here we have studied the cell biological and biophysical properties of receptors containing {alpha}6 and {beta}3 subunits by utilizing fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently-tagged {beta}3 subunits were fully functional compared to receptors with untagged {beta}3 subunits. We find that {beta}3 and {alpha}6-containing receptors are highly expressed in neurons, and co-localize with co-expressed, fluorescent {alpha}4 and {beta}2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of {beta}3 and {alpha}6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single {beta}3 subunit is incorporated into nAChRs containing this subunit, whereas multiple subunit stoichiometries exist for {alpha}4 and {alpha}6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Further, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of {alpha}6 and {beta}3-containing receptors using TIRF.
Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Mutagenesis/Chimeric approaches, Drug tolerance/dependence
7 October, 2007
Sowerby's Monograph of the genus Conus
Guido Poppe has made available as an eBook the Thesaurus Conchyliorum (1848-1847) from the Sowerby family. "This extremely rare book is very important in today’s nomenclature because of the many type figures. Only a few complete copies are still existing today and the antiquarian price for a whole set is extremely high. Today the complete work, all text pages and color plates are available for you to buy and download at moderate prices, probably less than at the time of its publication. You can buy the Thesaurus either complete, by volume or by family/genus."
Volume 3 of this 5 volume set includes Sowerby's "Monograph of the genus Conus" comprising 104 pages, 24 colour Plates containing 601 illustrations. This publication is available to download separately as a 33.5 Mb file in Adobe .pdb format. (Portrait of Sowerby III)
To purchase any of these historic and conchologically important volumes visit the CONCHOLOGY, INC web site at http://www.conchology.be Click on eBook, and Register or Log-In to purchase and download.
3 October, 2007
The Cone Collector #4
Antonio Monteiro has teamed with good friend Andre Poremski, who worked on the graphics, to give this essential publication for cone shell enthusiasts a professional touch.The new improved newsletter is currently available both in low resolution PDF format (1.8 Mb) and in high resolution PDF format (48 Mb) at the following sites www.theconecollector.net and www.seashell-collector.com
Articles include -
30 September, 2007
Contryphans from Conus amadis, Conus lorisii and Conus striatus
Thakur SS and Balaram P (2007). Rapid mass spectral identification of contryphans. Detection of characteristic peptide ions by fragmentation of intact disulfide-bonded peptides in crude venom. Rapid Commun Mass Spectrom. 21:3420-3426
Abstract: The mass spectrometric cleavage of intact disulfide-bonded peptides in Conus venom has been investigated. Contryphans containing a single disulfide bond are shown to fragment preferentially at X-Pro bonds, giving rise to linearized, unsymmetrical cystine peptides, which subsequently fragment by multiple pathways
at the disulfide bridge. Cleavage at the disulfide bond can be initiated by initial loss of the C(alpha)H or C(beta)H proton, resulting in distinct product ions, with the subsequent loss of elemental sulfur, H(2)S or H(2)S(2). Contryphans from Conus amadis, Conus loroisii, and Conus striatus are presented as examples, in which detailed assignment of the product ions resulting from tandem mass spectrometric analysis of the intact disulfide is also accomplished. Characteristic fragments arising from conserved contryphan sequences can be used
as diagnostic, permitting rapid identification of this class of peptides in crude venom. The observed fragment ions obtained for contryphans in diverse cone snail species are also compared. Copyright (c) 2007 John Wiley & Sons, Ltd.
12 September, 2007
Conotoxin MII recognizes alpha6-containing nicotinic receptors
Grady SR, Salminen O, Laverty DC, Whiteaker P, McIntosh JM, Collins AC and Marks
MJ. (2007) The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of
mouse striatum. Biochem Pharmacol. 2007 Jul 27; [Epub ahead of print]
Abstract: This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and alpha-conotoxin MII (alpha-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the alpha6 subunit (alpha4alpha6beta2beta3, alpha6beta2beta3, alpha6beta2) and bind alpha-CtxMII with high affinity. One of these three subtypes (alpha4alpha6beta2beta3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for alpha-CtxMII (alpha4beta2, alpha4alpha5beta2)are somewhat more numerous than the alpha6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.
What is the pain target for alpha-conotoxins ?
Nevin ST, Clark RJ, Klimis H, Christie MJ, Craik DJ, Adams DJ. (2007) Are {alpha}9{alpha}10 nicotinic acetylcholine receptors a pain target for {alpha}-conotoxins ? Mol Pharmacol. 2007 Sep 5; [Epub ahead of print]
Abstract: The synthetic alpha-conotoxin Vc1.1 (ACV1) is a small disulfide bonded peptide
currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the
native post-translationally-modified peptide vc1a does not act as an analgesic in
vivo in rat models of neuropathic pain. Recently, it has been proposed that the
primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor
(nAChR). We show that Vc1.1 and its post-translationally modified analogues vc1a,
[P6O]Vc1.1 and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked
currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10
nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1
for the treatment of neuropathic pain.
Conus: a source of selective sodium channel blockers for pain treatment
Cummins TR, Sheets PL and Waxman SG. (2007) The roles of sodium channels in nociception: Implications for mechanisms of pain. Pain 131: 243-257. .
Abstract: Understanding the role of voltage-gated sodium channels in nociception may provide important insights into pain mechanisms. Voltage-gated sodium channels are critically important for electrogenesis and nerve impulse conduction, and a target for important clinically relevant analgesics such as lidocaine. Furthermore, within the last decade studies have shown that certain sodium channel isoforms are predominantly expressed in peripheral sensory neurons associated with pain sensation, and that the expression and functional properties of voltage-gated sodium channels in peripheral sensory neurons can be dynamically regulated following axonal injury or peripheral inflammation. These data suggest that specific voltage-gated sodium channels may play crucial roles in nociception. Experiments with transgenic mice lines have clearly implicated Na(v)1.7, Na(v)1.8 and Na(v)1.9 in inflammatory, and possibly neuropathic, pain. However the most convincing and perhaps most exciting results regarding the role of voltage-gated sodium channels have come out recently from studies on human inherited disorders of nociception. Point mutations in Na(v)1.7 have been identified in patients with two distinct autosomal dominant severe chronic pain syndromes. Electrophysiological experiments indicate that these pain-associated mutations cause small yet significant changes in the gating properties of voltage-gated sodium channels that are likely to contribute substantially to the development of chronic pain. Equally exciting, recent studies indicate that recessive mutations in Na(v)1.7 that eliminate functional current can result in an apparent complete, and possibly specific, indifference to pain in humans, suggesting that isoform specific blockers could be very effective in treating pain. In this review we will examine what is known about the roles of voltage-gated sodium channels in nociception.
[The uO-conotoxin MrVIB from Conus marmoreus has been shown to be 6- to 10-fold more potent at blocking Nav1.8 currents than neuronal TTX-sensitive currents and when infused intrathecally into mice produced substantial analgesia at doses that showed much less severe motor side-effects than lidocaine (Bulaj et al., 2006; Ekberg et al., 2006). In addition, a small molecule inhibitor of Nav1.8 channels, termed A-803467, appears to be both a potent and highly selective inhibitor of Nav1.8 channels. It was able to block spontaneous and evoked action potentials in rat sensory neurons and showed efficacy in a wide range of animal pain models, including models of acute mechanical pain, inflammatory pain and neuropathic pain (Jarvis et al., 2007). These studies suggest that Nav1.8 selective antagonists may have better therapeutic indices for treating pain than the currently available nonselective antagonists of voltage-gated sodium channels].
Other subtype-selective sodium channel blockers have been identified in Conus kinoshitai (conotoxin KIIIA, Zhang et al 2007), Conus striatus (conotoxin SIIIA, Green et al 2007), Conus consors (mu-conotoxin CnIIIA & CnIIIB, Zhang et al 2006), Conus catus (mu-conotoxin CIIIA, Zhang et al 2006), and Conus magus (mu-conotoxin MIIIA, Zhang et al 2006) .
See also: Cummins, TR and Rush AM (2007) Voltage-gated sodium channel blockers for the treatment of neuropathic pain. Expert Rev Neurother. 7:1597-1612.
6 September, 2007
Marine sourced compounds for neurology
Martinez A. (2007) Marine-derived drugs in neurology.
Curr Opin Investig Drugs. 8: 525-530. Review.
Abstract:The oceans provide a rich source of structurally unique compounds that have
demonstrated significant biological activities in a range of indications. In
particular, the development of marine compounds is emerging as an important field
for neurology. Several marine-derived compounds are currently in clinical trials
or have been launched for the treatment of neuropathic pain, schizophrenia and
Alzheimer's disease. This review describes the development of several of these
compounds, specifically covering the conopeptides, anabaseine and omega-3 fatty
acids for the potential treatment of various neurological disorders.
Protein disulfide isomerase from Conus marmoreus
Wang ZQ, Han YH, Shao XX, Chi CW, Guo ZY.(2007) Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus. FEBS J. 274: 4778-4787.
Abstract:The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.
30 August, 2007
Phase II clinical trial of conotoxin MVIIA (Ziconotide/Prialt) combined with intrathecal morphine
Wallace MS, Kosek PS, Staats P, Fisher R, Schultz DM and Leong M. (2007) Phase II, open-label, multicenter study of combined intrathecal morphine and Ziconotide: Addition of Ziconotide in patients receiving intrathecal morphine for severe chronic pain. Pain Medicine, OnlineEarly ArticlesPublished article online: 28-Aug-2007
Abstract: OBJECTIVE:. To assess the safety and efficacy of adding intrathecal ziconotide to intrathecal morphine in patients being treated with a stable intrathecal morphine dose. DESIGN: Phase II, multicenter, open-label study with a 5-week titration phase and an extension phase. SETTING: Outpatient clinics. PATIENTS: Patients with suboptimal pain relief receiving stable intrathecal morphine doses (2–20 mg/day). INTERVENTIONS: Intrathecal morphine dosing remained constant during the titration phase. Ziconotide therapy began at 0.60 µg/day and was titrated to a maximum of 7.2 µg/day. During the extension phase, ziconotide and intrathecal morphine dosing were adjusted at the investigator’s discretion. OUTCOME MEASURES: Safety was assessed primarily via adverse event reports. Efficacy was analyzed via percentage change on the visual analog scale of pain intensity and in weekly systemic opioid consumption. RESULTS: Twenty-six patients were enrolled. Treatment-emergent adverse events were generally mild or moderate; the most common (?15% of patients in either study phase) study drug-related (i.e., ziconotide/morphine combination [or ziconotide monotherapy in the extension phase only]) events were confusion, dizziness, abnormal gait, hallucinations, and anxiety. The mean percentage improvement in visual analog scale of pain intensity scores was 14.5% (95% confidence interval: ?9.4% to 38.5%) from baseline to week 5 and varied during the extension phase (range: ?0.4% to 42.8%). Mean percentage change from baseline in systemic opioid consumption was ?14.3% at week 5 and varied considerably during the extension phase. CONCLUSIONS: Ziconotide, combined with stable intrathecal morphine, may reduce pain and decrease systemic opioid use in patients with pain inadequately controlled by intrathecal morphine alone.
29 August, 2007
An analgesic peptide mu-conotoxin KIIIA from Conus kinoshitai irreversibly blocks neuronal sodium channels
Zhang M-M, Green BR, Catlin P, Fiedler B, Azam L, Chadwick A, Terlau H, McArthur JR, French RJ, Gulyas J, Rivier JE, Smith BJ, Norton RS, Olivera BM, Yoshikami D and Bulaj G. (2007) Structure/function characterization of mu -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian sodium channels. J. Biol. Chem. 282: 30699-30706.
Abstract: Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. u-conopeptides previously characterized as inhibitors of TTX-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ~20% of the TTX-resistant sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and and NaV1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Nav1.2 and only partially for Nav1.6. Other isoforms were blocked reversibly: Nav1.3 (IC50, 8uM) and Nav1.4 (IC50, 80 nM). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4: replacement of Trp8 resulted in reversible block of Nav1.2, whereas replacement of Lys7, Trp8, or Asp11, yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Nav1.2, and that further engineering of mu-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.
26 August, 2007
Convection-enhanced delivery (CED) of omega-conotoxins MVIIA and GVIA provides prolonged seizure protection in rats.
Gasior M, White N, Rogawski MA. (2007) Prolonged attenuation of amygdala-kindled seizure measures in rats by convection-enhanced delivery of the N-type calcium channel antagonists {omega}-Conotoxin GVIA and {omega}-Conotoxin MVIIA. J Pharmacol Exp Ther. 2007 Aug 23; [Epub ahead of print]
Abstract: Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA can attenuate kindling measures in fully amygdala kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-conotoxin GVIA (0.005, 0.05, 0.5 nmol), omega-conotoxin MVIIA (0.05, 0.15, 0.5 nmol), proteolytically inactivated omega-conotoxin MVIIA (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-conotoxin GVIA and omega-conotoxin MVIIA over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h post infusion and then gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-conotoxin MVIIA had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high molecular weight presynaptic N-type calcium channel blockers can produce long lasting inhibition of brain excitability and may provide prolonged seizure protection in focal seizure disorders.
Selective inhibition of alpha-CtxMII-sensitive nAChRs may counter ethanol reinforcement clues
Lof E, Olausson P, Debejczy A, Stomberg R, McIntosh JM, Taylor JR, Soderpalm B. (2007)Nicotinic acetylcholine receptors in the ventral tegmental area mediate the dopamine activating and reinforcing properties of ethanol cues. Psychopharmacology (Berl). 2007 Aug 17; [Epub ahead of print]
Abstract: RATIONALE: Cues associated with alcohol can elicit craving, support drug-seeking and precipitate relapse. OBJECTIVES: We investigated the possible involvement of nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA) in the conditioned reinforcing properties of ethanol-associated stimuli in the rat. MATERIALS AND METHODS: First, using in vivo microdialysis, we analyzed the effect of VTA perfusion of the nonselective nAChR antagonist mecamylamine (MEC) or the selective alpha4beta2* nAChR antagonist dihydro-beta-erythroidine (DHbetaE) on the nucleus accumbens (nAc) dopaminergic response to the presentation of an ethanol-associated conditioned stimulus (CS). Second, rats were trained to associate a tone + light CS with the presentation of 10% ethanol and were subsequently tested on the acquisition of a new instrumental response with conditioned reinforcement (CR) after local VTA infusion of MEC, DHbetaE, or alpha-Conotoxin MII (alpha-CtxMII, a selective alpha3beta2* and alpha6* nAChR antagonist). RESULTS: The ethanol-associated CS elevated nAc dopamine, an effect that was blocked by VTA perfusion of MEC but not DHbetaE. Systemic administration of MEC or local VTA infusion of MEC or alpha-CtxMII selectively blocked ethanol-associated CR, whereas systemic DHbetaE had no effect. CONCLUSIONS: We hypothesize a novel mechanism by which alcohol-associated cues promote drug-seeking behavior via activation of dopamine-stimulating alpha-CtxMII-sensitive nAChRs in the VTA. Pharmacological manipulations of selective nAChRs may thus be possible treatment strategies to prevent cue-induced relapse.
Slow folding of Conantokin-T from Conus tulipa
Du D, Bunagan MR, Gai F. (2007) The effect of charge-charge interactions on the kinetics of {alpha}-helix formation. Biophys J. 93:4076-4082 Abstract: The formation of monomeric alpha-helix represents one of the simplest scenarios in protein folding; however, our current understanding of the folding dynamics of the alpha-helix motif is mainly based on studies of alanine-rich model peptides. To examine the effect of peptide sequence on the folding kinetics of alpha-helices, we studied the relaxation kinetics of a 21-residue helical peptide, Conantokin-T (Con-T), using time-resolved infrared spectroscopy in conjunction with a laser-induced temperature jump (T-jump) technique. Con-T is a neuroactive peptide found in the venom of the piscivorous cone snail Conus tulipa which contains a large number of charged residues. The T-jump relaxation kinetics of Con-T are distinctly slower than those of previously studied alanine-based peptides, suggesting that the folding time of alpha-helices is sequence dependent. Furthermore, it appears that the slower folding of Con-T can be attributed to the fact that its helical conformation is stabilized by charge-charge interactions or salt-bridges. While this finding contradicts an early molecular dynamics simulation, it also has implications for existing models of protein folding.
14 August, 2007
Metabolic Pharmaceuticals discontinues clinical trial program for neuropathic pain drug, ACV1 (conotoxin Vc1.1)
ASX Announcement. Melbourne 14 August 200 |
VISAYA November Vol. 2, No. 2 November 2007
PLENTY OF KNOTTIN. In a model of the knottin conotoxin gm9a, the protein backbone is shown as a smooth tube, and three disulfide bridges are shown in a ball-and-stick representation. In all knottins, a disulfide bridge (orange) penetrates a macrocycle (blue) formed by two other disulfides and interconnecting backbone segments. Although conotoxin gm9a's backbone is linear (noncyclic), the close proximity between its N- and C-termini shows how cyclic knottins can easily form by ring closure. MOLMOL [J. Mol. Graphics 14: 51 (1996)] Figure by Laurent Chiche (see 'Tying up Loose Ends' by Stu Borman 
